GFP Proton Shuttle Reaction IRC

Green fluorescent protein - Wikipedia

1 The chromophore within GFP is formed from a Ser-Tyr-Gly tripeptide in the primary protein structure by post-translational cyclodehydration and subsequent oxidation to yield the 4-( p -hydroxybenzylidene)imidazolinone (HO-BDI) moiety.

The green fluorescent protein ..

AB - Green fluorescent protein (GFP) has a chromophore that forms autocatalytically within the folded protein. Although many studies have focused on the precise mechanism of chromophore maturation, little is known about the kinetics of de novo chromophore maturation. Here we present a simple and efficient method for examining the de novo kinetics. GFP with an immature chromophore was synthesized in a reconstituted cell-free protein synthesis system under anaerobic conditions. Chromophore maturation was initiated by rapid dilution in an air-saturated maturation buffer, and the time course of fluorescence development was monitored. Comparison of the de novo maturation rates in various GFP variants revealed that some folding mutations near the chromophore promoted rapid chromophore maturation and that the accumulation of mutations could reduce the maturation rate. Our method will contribute to the design of rapidly maturing fluorescent proteins with improved characteristics for real-time monitoring of cellular events.

as it is the case with the synthesis of the GFP chromophore

The green fluorescent protein (GFP) from Aequorea victoria is a versatile reporter protein for monitoring gene expression and protein localization in a variety of cells and organisms. Despite many early successes using this reporter, wild type GFP is sub-optimal for most applications due to low fluorescence intensity when excited by blue light (488 nm), a significant lag in the development of fluorescence after protein synthesis, complex photoisomerization of the GFP chromophore and poor expression in many higher eukaryotes. To improve upon these qualities, we have combined a mutant of GFP with a significantly larger extinction coefficient for excitation at 488 nm with a re-engineered GFP gene sequence containing codons preferentially found in highly expressed human proteins. The combination of improved fluorescence intensity and higher expression levels yield an enhanced GFP which provides greater sensitivity in most systems.

The green fluorescent protein (GFP) chromophore is a ..

In green fluorescent protein (GFP), chromophore biosynthesis is initiated by a spontaneous main-chain condensation reaction. Nucleophilic addition of the Gly67 amide nitrogen to the Ser65 carbonyl carbon is catalyzed by the protein fold and leads to a heterocyclic intermediate. To investigate this mechanism, we substituted the highly conserved residues Arg96 and Glu222 in enhanced GFP (EGFP). In the R96M variant, the rate of chromophore formation is greatly reduced (time constant = 7.5 × 103 h, pH 7) and exhibits pH dependence. In the E222Q variant, the rate is also attenuated at physiological pH (32 h, pH 7) but is accelerated severalfold beyond that of EGFP at pH 9-10. In contrast, EGFP maturation is pH-independent and proceeds with a time constant of 1 h (pH 7-10). Mass spectrometric results for R96M and E222Q indicate accumulation of the pre-cyclization state, consistent with rate-limiting backbone condensation. The pH-rate profile implies that the Glu222 carboxylate titrates with an apparent pKa of 6.5 in R96M and that the Gly67 amide nitrogen titrates with an apparent pKa of 9.2 in E222Q. These data suggest a model for GFP chromophore synthesis in which the carboxylate of Glu222 plays the role of a general base, facilitating proton abstraction from the Gly67 amide nitrogen or the Tyr66 α-carbon. Arg96 fulfills the role of an electrophile by lowering the respective pKa values and stabilizing the α-enolate. Modulating the base strength of the proton-abstracting group may aid in the design of fast-maturing GFPs with improved characteristics for real-time monitoring of cellular events.

(GFP), chromophore biosynthesis is ..

TurboGFP can be expressed and detected in a wide range of organisms including cold-blooded animals. Mammalian cells transiently transfected with TurboGFP expression vectors produce bright fluorescence in 8-10 hrs after transfection. No cytotoxic effects or visible protein aggregation are observed. TurboGFP can be used in multicolor labeling applications with blue, true-yellow, red, and far-red fluorescent dyes.

PDB-101: Learning Resources: Green Fluorescent Protein (GFP)

The proposed, common, one-electron oxidized, radical intermediate for post-translation modifications in the GFP family has general implications for how proteins drive and control spontaneous post-translational chemical modifications in the absence of metal ions.

fluorescent protein chromophore synthesis revealed by ..

AB - In green fluorescent protein (GFP), chromophore biosynthesis is initiated by a spontaneous main-chain condensation reaction. Nucleophilic addition of the Gly67 amide nitrogen to the Ser65 carbonyl carbon is catalyzed by the protein fold and leads to a heterocyclic intermediate. To investigate this mechanism, we substituted the highly conserved residues Arg96 and Glu222 in enhanced GFP (EGFP). In the R96M variant, the rate of chromophore formation is greatly reduced (time constant = 7.5 × 103 h, pH 7) and exhibits pH dependence. In the E222Q variant, the rate is also attenuated at physiological pH (32 h, pH 7) but is accelerated severalfold beyond that of EGFP at pH 9-10. In contrast, EGFP maturation is pH-independent and proceeds with a time constant of 1 h (pH 7-10). Mass spectrometric results for R96M and E222Q indicate accumulation of the pre-cyclization state, consistent with rate-limiting backbone condensation. The pH-rate profile implies that the Glu222 carboxylate titrates with an apparent pKa of 6.5 in R96M and that the Gly67 amide nitrogen titrates with an apparent pKa of 9.2 in E222Q. These data suggest a model for GFP chromophore synthesis in which the carboxylate of Glu222 plays the role of a general base, facilitating proton abstraction from the Gly67 amide nitrogen or the Tyr66 α-carbon. Arg96 fulfills the role of an electrophile by lowering the respective pKa values and stabilizing the α-enolate. Modulating the base strength of the proton-abstracting group may aid in the design of fast-maturing GFPs with improved characteristics for real-time monitoring of cellular events.